乙型脑炎病毒E蛋白毒力关键位点的正向验证  

Forward verification of key virulence sites of Japanese encephalitis virus E protein

作　　者：范凤鸣 刘莉娜[1] 孙艳[1] 牟建超[1] 陈智[1] 杨欢 刘俐[1] 刘杰[1] 曾献武[1] 杨会强[1] FAN Feng-ming;LIU Li-na;SUN Yan;MU Jian-chao;CHEN Zhi;YANG Huan;LIU Li;LIU Jie;ZENG Xian-wu;YANG Hui-qiang(Chengdu Institute of Biological Products Co.Ltd.,Chengdu 610023,Sichuan Province,China)

机构地区：[1]成都生物制品研究所有限责任公司,四川成都610023

出　　处：《中国生物制品学杂志》2018年第11期1247-1252,共6页Chinese Journal of Biologicals

基　　金：重大新药创制科技重大专项(2018ZX09201006);863计划重大项目(2012AA02A401)

摘　　要：目的将乙型脑炎野毒株SA14囊膜蛋白(envelope,E)氨基酸残基107位点(E107)和138位点(E138)突变为乙型脑炎减毒株SA14-14-2的相应位点,正向分析两个氨基酸位点突变在乙型脑炎病毒(Japanese encephalitis virus,JEV)减毒中的作用。方法通过融合PCR方法分别扩增含有E107(1296-T)、E138(1389-A)、E107+138(1296-T/1389-A)突变位点的PCR片段,KasⅠ和BglⅡ酶切后替换乙型脑炎强毒SA14的嵌合病毒SA14-14-2/SA14的相应区域,构建3个突变株病毒的全长克隆,转染BHK21细胞获得突变株病毒。测定并比较3个突变株和嵌合病毒SA14-14-2/SA14、疫苗株SA14-14-2的蚀斑大小及细胞增殖特征、小鼠脑内神经毒力、小鼠皮下感染入脑能力,分析E107、E138位点突变对JEV的减毒作用。结果测序结果表明成功构建了3株突变株病毒,其中E107单位点突变对小鼠脑内神经毒力和皮下感染入脑能力无显著改变,蚀斑形态和细胞增殖能力也无明显变化。E138单位点突变及E138和E107双位点突变使病毒脑内神经毒力和皮下感染入脑能力急剧降低,其中E138和E107双位点突变使病毒皮下感染入脑能力完全消失,并且E138单位点突变以及E138和E107双位点突变病毒蚀斑较疫苗株SA14-14-2和嵌合病毒SA14-14-2/SA14明显偏小,但增殖能力有明显升高。结论 E138位点突变对JEV SA14的减毒起关键作用;在E138位点突变的前提下,E107位点与E138位点有显著的协同作用。Objective To verify the critical role of amino acid residues 107 and 138 in envelope protein（E107,E138）in attenuation of Japanese encephalitis virus by mutating these sites of wild virulent strain SA14 into the corresponding sites of attenuated strain SA14-14-2 by forward method. Methods The fragments containing E107（1296-T）,E138（1389-A）and E107＋138（1296-T/1389-T/A） mutation sites were amplified by overlap PCR,digested with KasⅠ and BglⅡ,with which the corresponding regions of chimeric virus SA14 14-2/SA14 were substituted. The full-length clones of three mutant viruses were constructed and transfected to BHK21 cells to obtain the mutant viruses. The roles of E107 and E138 site mutations in attenuation of JEV were analyzed by comparing the plaque sizes,proliferation characteristics in BHK21 cells as well as neurovirulence and neuroinvasiveness through subcutaneous route in mice of these mutant viruses,chimeric virus SA14 14-2/SA14 and attenuated virus strain SA14-14-2. Results Sequencing results showed that three mutant virus strains were successfully constructed. Single site mutation at E107 showed no significant effect on neurovirulence and neuroinvasiveness through subcutaneous route in mice as well as plaque morphology and proliferation ability in BHK21 cells of JEV. However,single site mutation at E138 and double site mutation of E107 and E138 significantly decreased the neurovirulence and neuroinvasiveness through subcutaneous route,especially,the double site mutation completely eliminated the neuroinvasiveness of JEV. The plaque size of JEV with single site mutation at E138 or double site mutation of E107 and E138 was significantly smaller,while the proliferation ability was significantly higher,than those of SA14-14-2 and SA14 14-2/SA14. Conclusion The mutation at E138 site plays an important role in the attenuation of JEV SA14. E107 and E138 sites showed significant synergistic effect on attenuation and characteristics of JEV.

关 键 词：乙型脑炎病毒 E蛋白 毒力关键位点 正向验证 减毒机制 

分 类 号：R373.31[医药卫生—病原生物学]